title: Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family.
abstract: BACKGROUND
  In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the "varietal character". Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown.
  
  
  RESULTS
  Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine.
  
  
  CONCLUSIONS
  Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.
authors.affiliation: Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa. mav@sun.ac.za.
authors.forename: Justin G
authors.initials: JG
authors.lastname: Lashbrooke
authors.forename: Philip R
authors.initials: PR
authors.lastname: Young
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Dockrall
authors.forename: Krishnan
authors.initials: K
authors.lastname: Vasanth
authors.forename: Melané A
authors.initials: MA
authors.lastname: Vivier
mesh_terms: D002338:Carotenoids / Q000378:metabolism; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology*; D027843:Vitis / Q000201:enzymology* / Q000378:metabolism
chemical_list: D002338:Carotenoids; D049308:Dioxygenases